Bottom Line:
MCM8-9 complex is required for homologous recombination (HR)-mediated repair of double-strand breaks (DSBs).MCM8-9 interacts with MRN and is required for the nuclease activity and stable association of MRN with DSBs.A cancer-derived point mutation or an SNP on MCM8 associated with premature ovarian failure (POF) diminishes the functional activity of MCM8.

ABSTRACTMCM8-9 complex is required for homologous recombination (HR)-mediated repair of double-strand breaks (DSBs). Here we report that MCM8-9 is required for DNA resection by MRN (MRE11-RAD50-NBS1) at DSBs to generate ssDNA. MCM8-9 interacts with MRN and is required for the nuclease activity and stable association of MRN with DSBs. The ATPase motifs of MCM8-9 are required for recruitment of MRE11 to foci of DNA damage. Homozygous deletion of the MCM9 found in various cancers sensitizes a cancer cell line to interstrand-crosslinking (ICL) agents. A cancer-derived point mutation or an SNP on MCM8 associated with premature ovarian failure (POF) diminishes the functional activity of MCM8. Therefore, the MCM8-9 complex facilitates DNA resection by the MRN complex during HR repair, genetic or epigenetic inactivation of MCM8 or MCM9 are seen in human cancers, and genetic inactivation of MCM8 may be the basis of a POF syndrome.

f2: ssDNA generation at DSB depends on MCM8 or MCM9.(a) ssDNA foci measured by BrdU staining without DNA denaturation and γH2AX immunofluorescence foci. Representative images on left and quantification of foci-positive cells on the right. Scale bar, 10 μm. ***P<0.005, **P<0.01; Student's t-test. (b) ssDNA foci (BrdU foci) in MEFs with WT MCM9 (WT/WT) or with homozygous mutation for MCM9 (XG/XG) after exposure to cisplatin. Bottom: mean±s.d. of triplicates. Scale bar, 10 μm. ***P<0.005; Student's t-test. (c) Quantitative measurement of DNA resection 4 h after addition of 4-OHT to ER-AsiSI U2OS cells. Percentage of ssDNA at indicated sites was measured by qPCR using the primer pairs indicated on the cartoon after digestion with BsrGI. Bottom: % of ssDNA at different sites. ***P<0.005, *P<0.05; Student's t-test. All error bars represent s.d. of the mean from triplicates.

Mentions:
To directly examine whether MCM8-9 is required for ssDNA generation, the generation of ssDNA at DSB sites was directly visualized by immunostaining with anti-BrdU (5-bromodeoxyuridine) antibody without denaturation of the double-stranded DNA after labelling of genomic DNA with BrdU in a previous cell cycle. Cisplatin increased the number of cells with BrdU signal, but this was decreased by depletion of MCM8 or MCM9 (Fig. 2a). Similarly, cisplatin treatment of mouse embryonic fibroblast (MEF) cells from Mcm9- (XG/XG) mice produced fewer ssDNA-positive cells compared with wild-type (WT) MEFs (Fig. 2b). Cell cycle profile of the MEF cells from XG/XG mice was not changed by the 4-h cisplatin treatment, suggesting that the decrease in ssDNA formation could not be explained by a change in S phase progression in these cells (Supplementary Fig. 2). Next, we utilized a new assay for quantitatively measuring ssDNA at regions adjoining a specific DSB site induced by the restriction enzyme AsiSI in the estrogen receptor (ER)-AsiSI U2OS cells22. The formation of ssDNA makes BsrG1 sites in the adjoining DNA resistant to digestion by that enzyme, thus allowing us to measure whether the end resection complex has digested past each of the three BsrGI site. DSB induced by 4-hydroxytamoxifen (4-OHT) treatment resulted in more ssDNA at the BsrG1 site closer to AsiSI cut site, but at both this site and the next, depletion of MCM8 or MCM9 decreased the generation of ssDNA (Fig. 2c). Depletion of MRE11 also affected ssDNA formation at these sites, validating the assay. Therefore MCM8-9 is required for ssDNA formation at DSBs before HR.

f2: ssDNA generation at DSB depends on MCM8 or MCM9.(a) ssDNA foci measured by BrdU staining without DNA denaturation and γH2AX immunofluorescence foci. Representative images on left and quantification of foci-positive cells on the right. Scale bar, 10 μm. ***P<0.005, **P<0.01; Student's t-test. (b) ssDNA foci (BrdU foci) in MEFs with WT MCM9 (WT/WT) or with homozygous mutation for MCM9 (XG/XG) after exposure to cisplatin. Bottom: mean±s.d. of triplicates. Scale bar, 10 μm. ***P<0.005; Student's t-test. (c) Quantitative measurement of DNA resection 4 h after addition of 4-OHT to ER-AsiSI U2OS cells. Percentage of ssDNA at indicated sites was measured by qPCR using the primer pairs indicated on the cartoon after digestion with BsrGI. Bottom: % of ssDNA at different sites. ***P<0.005, *P<0.05; Student's t-test. All error bars represent s.d. of the mean from triplicates.

Mentions:
To directly examine whether MCM8-9 is required for ssDNA generation, the generation of ssDNA at DSB sites was directly visualized by immunostaining with anti-BrdU (5-bromodeoxyuridine) antibody without denaturation of the double-stranded DNA after labelling of genomic DNA with BrdU in a previous cell cycle. Cisplatin increased the number of cells with BrdU signal, but this was decreased by depletion of MCM8 or MCM9 (Fig. 2a). Similarly, cisplatin treatment of mouse embryonic fibroblast (MEF) cells from Mcm9- (XG/XG) mice produced fewer ssDNA-positive cells compared with wild-type (WT) MEFs (Fig. 2b). Cell cycle profile of the MEF cells from XG/XG mice was not changed by the 4-h cisplatin treatment, suggesting that the decrease in ssDNA formation could not be explained by a change in S phase progression in these cells (Supplementary Fig. 2). Next, we utilized a new assay for quantitatively measuring ssDNA at regions adjoining a specific DSB site induced by the restriction enzyme AsiSI in the estrogen receptor (ER)-AsiSI U2OS cells22. The formation of ssDNA makes BsrG1 sites in the adjoining DNA resistant to digestion by that enzyme, thus allowing us to measure whether the end resection complex has digested past each of the three BsrGI site. DSB induced by 4-hydroxytamoxifen (4-OHT) treatment resulted in more ssDNA at the BsrG1 site closer to AsiSI cut site, but at both this site and the next, depletion of MCM8 or MCM9 decreased the generation of ssDNA (Fig. 2c). Depletion of MRE11 also affected ssDNA formation at these sites, validating the assay. Therefore MCM8-9 is required for ssDNA formation at DSBs before HR.

Bottom Line:
MCM8-9 complex is required for homologous recombination (HR)-mediated repair of double-strand breaks (DSBs).MCM8-9 interacts with MRN and is required for the nuclease activity and stable association of MRN with DSBs.A cancer-derived point mutation or an SNP on MCM8 associated with premature ovarian failure (POF) diminishes the functional activity of MCM8.

ABSTRACTMCM8-9 complex is required for homologous recombination (HR)-mediated repair of double-strand breaks (DSBs). Here we report that MCM8-9 is required for DNA resection by MRN (MRE11-RAD50-NBS1) at DSBs to generate ssDNA. MCM8-9 interacts with MRN and is required for the nuclease activity and stable association of MRN with DSBs. The ATPase motifs of MCM8-9 are required for recruitment of MRE11 to foci of DNA damage. Homozygous deletion of the MCM9 found in various cancers sensitizes a cancer cell line to interstrand-crosslinking (ICL) agents. A cancer-derived point mutation or an SNP on MCM8 associated with premature ovarian failure (POF) diminishes the functional activity of MCM8. Therefore, the MCM8-9 complex facilitates DNA resection by the MRN complex during HR repair, genetic or epigenetic inactivation of MCM8 or MCM9 are seen in human cancers, and genetic inactivation of MCM8 may be the basis of a POF syndrome.